In order to better understand and define the role, nature, interrelationships and origin of circulating species of plasminogen activator (PA[s]) in the blood, it is essential to systematically characterize the different molecular forms of zymogen and active PA forms produced by various cell types in close association with the blood and to determine whether these PA forms are related to urokinase (u-PA) and/or tissue PA (t-PA). The overall goal of this study is to isolate and purify each of the major multiple molecular forms (zymogen/enzyme) of u-PA-type (M(r) 54K, 90K) and t-PA-type PAs (Mr 54K, 70K, 110K), produced/secreted by cultured human umbilical vein endothelial cells (HUVEC[s]), and to characterize these PA forms in terms of their physicochemical, enzymatic and immunochemical properties. HUVEC-produced PA forms (zymogen/-enzyme) will be isolated and purified from 96 hr serum-free conditioned media by a combination of methods; benzamidine-Sepharose 4B affinity chromatography, Sephacryl S-200 gel filtration, and affinity chromatography on either specific anti-u-PA IgG-Sepharose 4B or specific anti-t-PA IgG-Sepharose 4B. Purified PAs (zymogen/enzyme) will be characterized in terms of: molecular weight and subunit composition; active sites; pI's; peptide maps; amino acid compositions and N-terminal sequence analysis; s(20 w) and Stoke's radius; peptidase and esterase activities; effect of inhititors on PA activity; characteristics of human plasminogen activation (k(cat), K(m) and k(cat)/K(m)); and immunological cross-reactivity with specific anti-u-PA IgG (including monoclonal antibodies), specific anti-t-PA IgG and monoclonal antibodies prepared against each of the HUVEC-derived PAs. In addition, we will also examine the mechanism of conversion of purified PA zymogens to enzymes by treatment with serine proteases. Methods include: affinity and gel filtration chromatography; radiolabeling; SDS-PAGE; autofluorography; isoelectrofocusing; HPLC; sequence analysis; analytical ultracentrifugation; and enzyme and IRMA assays. These studies will define the specific properties, interrelationships and nature of active and zymogen forms of HUVEC-produced PAs and should provide some insight into the origin of circulating PAs and their respective roles in EC-function, EC-mediated fibrinolysis and hence the maintenance of vascular patency.